The present invention pertains to fluorescent optical imaging systems and, more particularly, to a non-confocal fluorescence imaging system for broad scale imaging of relatively large samples.
The present invention relates to the simultaneous imaging of two or more fluorescently-labeled samples in a scanning optical microscope. The field of view obtained with this system is substantially larger than conventional fluorescence microscopes, in which the field of view is typically limited by the optical design of the objective lens. This invention can be applied to, but is not limited to, samples such as DNA microarrays or tissue microarrays, where short depth of focus is not required, and, in fact, would degrade system performance (Cheung, V. G., M. Morley, F. Aguilar, A. Massimi, R. Yucherlapati and G. Childs, xe2x80x9cMaking and reading microarrays,xe2x80x9d Nature Genetics Supplement 21:15-19(1999)). It is also suitable for samples that implement fluorescent labels with small Stokes shifts and/or overlapping absorption and emission spectra.
Difficulties can arise in fluorescence microscopy when imaging multiple fluors with close spectral properties. It can be impractical to excite only one fluor with a source (e.g. laser) beam due to the overlap of absorption spectra or the spectral bandwidth of the source. The spectral emission ranges from multiple fluors may overlap, making it difficult to direct the emission from each fluor efficiently to a single detector, without crosstalk. Even if the emission ranges don""t overlap, they may be close enough to make it difficult to obtain an effective optical component (e.g. filter, grating, or prism) for separating them. One solution is to scan each wavelength independently, and then assemble a composite image from multiple scans. However, speed and image registration become issues in this case.
U.S. Pat. No. 5,304,810 of Amos discloses a scanning confocal microscope where two or more source beams with different angular orientations illuminate two distinct spots on a sample located in the object plane of a microscope objective. The resulting reflected or fluorescent light is detected by an equal number of spaced detectors, each one receiving light from a single illuminated spot. With this system, the region from which light is collected by each detector (its xe2x80x9cfield of viewxe2x80x9d) is spatially limited to nearly the same area as the excitation spot size.
An advantage of the system of Amos is that it achieves high spatial resolution at each distinct point illuminated on the specimen, which for many imaging applications is highly desirable. However, for other applications, a lower resolution image suffices.
Shalon, D., S. Smith and P. O. Brown, xe2x80x9cA DNA micro-array system for analyzing complex DNA samples using two-color fluorescent probe hybridization,xe2x80x9d Genome Research 6:639-645 (1996) describe a scanner for dual wavelength fluorescence detection of DNA microarrays that illuminates sizable spots on the sample. This is accomplished by intentionally underfilling the objective entrance pupil (i.e. the back aperture), which, by reducing the numerical aperture (NA) of the converging beam, increases the diffraction limited spot size in the focal plane. Note that substantially underfilling the objective aperture with a single-transverse-mode laser beam likely results in a Gaussian intensity distribution in the focal plane, whereas overfilling the objective aperture, as is often done in laser scanning microscopy, produces a distribution in the focal plane that approaches an Airy function.
As is well known in the field, it is possible to improve the axial resolution (reduce the depth of focus) of an optical microscope by implementing it as a confocal design. The essential benefit of a confocal microscope is the rejection of light from out-of-focus planes, allowing imaging of thick samples without blurring (Corle, T and G. Kino, Confocal Scanning Optical Microscopy and Related Imaging Systems, Academic Press, San Diego 1996). Cheung et al. (1999) observed that a confocal configuration actually reduced the signal-to-noise ratio, and was therefore not beneficial, in scanning microarrays. Furthermore, the depth-of-focus produced in a high numerical aperture confocal system is substantially less than the typical flatness of a microscope slide. This can also be an issue in a non-confocal high NA system, but is more readily overcome. For example, in the present invention low NA source beams are combined with large area detectors to reduce the sensitivity to defocus.
U.S. Pat. No. 5,459,325 of Heuton and Van Gelder discloses a high-speed fluorescence scanner that implements a light weight scan head containing a lens and mirror. This design has the advantage of variable field of view. However, it relies on a spectral dispersion device for separating the excitation and emission beams. As discussed above, there are practical obstacles to spectral beamsplitting that limit its flexibility in some applications. Thus, an efficient, multi-wavelength scanning system for measurement of samples that do not benefit from strict depth discrimination is needed. Furthermore, it should overcome the limitations of spectral beamsplitting to allow free use of available fluors. The present invention is directed at providing a solution to this problem.
The fluorescent optical imaging system of the present invention, originally designed for the purpose of imaging hybridized DNA chips, has a wide range of potential capabilities. A first aspect of the imaging system of the present invention comprises an optical source for generating at least two excitation beams with spatial separation for illuminating on a sample at least two distinct illuminated spots that are spaced apart a predetermined distance, with the illuminated spots generating at least two emission beams spatially or angularly separated, a detector for receiving each emission beam, and an objective element for directing the excitation beams onto the sample. Each detector has a field of view (receives light from a region) on the sample that is larger than an illuminated spot, but encompasses only a single illuminated spot.
According to an aspect of the invention, the objective element includes a scanning mechanism for directing the excitation beams onto an area of the sample. Preferably, the scanning mechanism includes means for moving the objective element in a first direction. With this embodiment, the system further comprises means for moving the sample in a second, typically perpendicular direction. Data processing controls and suitable imaging techniques are used to create an image of a scanned sample.
According to another aspect of the invention, the optical source and the objective element generate the illuminated spots in a manner creating spots that are relatively large spots as compared to diffraction limited spots of a moderate to high numerical aperture (NA) microscope objective, such as typically used in a confocal microscope. This is an important feature of one aspect of the invention, and is discussed in more detail herein.
According to another aspect of the invention, there is spatial separation of the two excitation beams. Preferably, the excitation beams are angularly offset with respect to each other. In addition, the system further comprises means for spatially separating the emission beams and redirecting the emission beams, each towards their own respective detector. Spatial separation of the excitation and emission beams is achieved, preferably, by means of a mirror with a small optical hole. However, other designs are possible, such as a small mirror that is smaller than an emission beam, or a prism.
According to another aspect of the invention, each detector is displaced from a focal point of its respective emission beam. This provides a degree of de-focus, which allows for broader imaging techniques, as discussed herein.
A second aspect of the imaging system of the present invention comprises an optical source for generating an excitation beam to be directed at a sample to be imaged in a manner generating an emission beam from the sample, a detector for receiving the emission beam from the sample, an objective element between the optical source and the sample for directing the excitation beam onto the sample and for receiving the emission beam from the sample in a manner where the excitation beam and emission beam at least partially occupy the same space, and an optical element for geometrically separating the excitation beam from the emission beam and directing the emission beam towards the detector. At the point of separation of the two beams, the excitation beam partially occupies the emission beam.
According to an aspect of this embodiment of the imaging system, the excitation beam occupies a part of the objective element and the emission beam occupies substantially all of the objective element. Preferably, the objective element is a lens, however, a parabolic mirror could also be used, as well as a number of other dioptric, catoptic, and catadioptric imaging systems.
According to another aspect of the invention, the optical element includes a mirror with a small hole. Alternative designs for the optical element, also referred to as a beamsplitter herein, include a small mirror that is smaller than an emission beam, a prism, and several other designs as described below.
According to another aspect of the invention, the excitation beam occupies a small percentage of the space occupied by the emission beam.
According to yet another aspect of the invention, the optical source is adapted to generate first and second excitation beams to be directed by the objective element toward the sample in a manner generating first and second emission beams. Preferably, the first and second excitation beams are angularly displaced from each other. Alternatively, however, the first and second excitation beams may be parallel to each other. For this alternative design, the objective element may include first and second objective lenses, one for each excitation beam.